1-Propanaminium, 3-[[9,10-dihydro-4-(methylamino)-9,10-dioxo-1-anthracenyl]amino]-N,N-dimethyl-N-propyl-, bromide (1:1)

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-06-06 to 2007-08-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Similar to OECD 414 with the following deviations: (1) the test substance was administered to pregnant animals from gestation day 6 to day 20 rather than at least from implantation to one day prior to the day of scheduled kill.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

See rationale for reliability above.

Principles of method if other than guideline:

N/A

GLP compliance:

yes (incl. QA statement)

Remarks:

N/A

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

481-170-7

EC Name:

-

Cas Number:

502453-61-4

Molecular formula:

Hill formula: C23H30BrN3O2 CAS formula: C23H30N3O2.Br

IUPAC Name:

dimethyl(3-{[4-(methylamino)-9,10-dioxo-9,10-dihydroanthracen-1-yl]amino}propyl)propylazanium bromide

Constituent 2

Reference substance name:

Dimethyl-(3-(4-methylamino-9,10-dioxo-9,10-dihydro-anthracen -1-ylamino)propyl)propylammonium bromide

IUPAC Name:

Dimethyl-(3-(4-methylamino-9,10-dioxo-9,10-dihydro-anthracen -1-ylamino)propyl)propylammonium bromide

Details on test material:

- Name of test material (as cited in study report): B119 HC Blue 16
- Molecular formula (if other than submission substance): C23H30N3O2Br
- Molecular weight (if other than submission substance): 460.42
- Smiles notation (if other than submission substance): N/A
- InChl (if other than submission substance): N/A
- Structural formula attached as image file (if other than submission substance): N/A
- Substance type: Active
- Physical state: solid, dark blue powder

Test animals

Species:

rat

Strain:

other: HanRcc:WIST (SPF Quality)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd Laboratory Animal Services, Wolferstrasse 4, CH-4414 Fullinsdorf/Switzerland
- Age at study initiation: 10 weeks at pairing
- Weight at study initiation: 190-227 grams (day 0 post coitum)
- Fasting period before study: N/A
- Housing: Animals were housed individually in Makrolon type-3 cages with wire mesh tops and standardized granulated softwood bedding.
- Diet (e.g. ad libitum): Pelleted standard Kliba-Nafag 3433 rat/mouse maintenance diet was available ad libitum.
- Water (e.g. ad libitum): Community tap water from Fullinsdorf in bottles was available ad libitum.
- Acclimation period: Seven days prior to pairing under test conditions with an evaluation of the health status


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12 with background music played at a centrally defined low volume for at least 8 hours during the light period.


IN-LIFE DATES: From: 2006-06-20 To: 2006-07-12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: highly purified water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dose formulations were prepared in terms of material as supplied by the Sponsor. The test substance was weighed into a glass beaker on a tared precision balance and the vehicle was added (w/v). Using an appropriate magnetic stirrer or homogenizer a homogeneous mixture was prepared. Separate formulations were prepared for each concentration. As the pH was measured appropriately of each preparation of the dose formulations (with values between 47.9 and 6.5) it was not considered necessary, that the pH needed to be adjusted by addition of HCl/NaOH. The vehicle without test substance was noted with a pH between 5.2 and 6.2. During the administration period, homogeneity of the test substance in the vehicle was maintained using a magnetic stirrer. Dose formulations were prepared daily and stored at room temperature (15-25 degrees C) in glass beakers.


DIET PREPARATION
- Rate of preparation of diet (frequency): N/A
- Mixing appropriate amounts with (Type of food): N/A
- Storage temperature of food: N/A


VEHICLE
- Justification for use and choice of vehicle (if other than water): N/A
- Concentration in vehicle: N/A
- Amount of vehicle (if gavage): 10 mL/kg bw with a daily adjustment of the individual volume to the actual body weight.
- Lot/batch no. (if required): N/A
- Purity: N/A

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for determination of concentration, homogeneity and stability (4 hours and 7 days) of the dose formulations were taken during the first week of the administration period. Additionally, samples for determination of concentrations and homogeneity were taken during the last week of the administration period.
On each occasion three samples of approximately 2 g were taken from the top, middle and bottom of each formulation and transferred into flat bottomed flasks. Stability samples were taken from the middle only. The samples were frozen (-25 to -15 degrees C) pending analysis. Samples were sent on dry ice to Dr. D. Flade, RCC Ltd, Environmental Chemistry & Pharmanalytics, CH-4452 Itingen/Switzerland. Analysis was performed using a method provided by the Sponsor.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: After acclimatization, females were housed with sexually mature males (1:1) in special automatic mating cages, i.e. with synchronized timing to initiate the nightly mating period. This system reduced the variation in the copulation times of the different females.
- Length of cohabitation: Animals were cohoused until evidence of copulation was observed.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: N/A
- Further matings after two unsuccessful attempts: No
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: No

Duration of treatment / exposure:

Animals were treated from day 6 post coitum (implantation) through to day 20 post coitum (last treatment)

Frequency of treatment:

Animals were treated daily in the morning.

Duration of test:

Animals were sacrificed on day 21 post coitum.

No. of animals per sex per dose:

22 mated females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on a dose range-finding study (RCC Study No. A62976), where the animal group administered with high dose of 1000 mg/kg did not show any toxic effects.
- Rationale for animal assignment: Rats were assigned to the different groups using a computer generated random algorithm
- Other: N/A

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily
- Cage side observations included: All animals were checked for mortalities. Any female sacrificed or found dead during the study was subjected to macroscopic examination with emphasis on the uterus and its contents. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4 % formaldehyde solution. In addition, all animals were observed for signs of reaction to treatment and/or symptoms of ill health.


DETAILED CLINICAL OBSERVATIONS: No
- Time schedule: N/A


BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded daily from day 0 until day 21 post coitum.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes-food consumption; no-compound intake
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes; measured on 3 day intervals from day 0 to 3, 3 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18 and 18 to 21 of gestation.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: N/A


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): N/A
- Time schedule for examinations: N/A


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: all internal organs


OTHER: N/A

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes; The uteri (and contents) of all females with live fetuses were weighed at necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain.
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: position of fetus in the uterus

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: at least half per litter
- Skeletal examinations: Yes: any animals not used for soft tissue or head examinations
- Head examinations: Yes: at least half per litter

Statistics:

The following statistical methods were used to analyze body weights, food consumption, reproduction and skeletal examination data:
-Means and standard deviations of various data were calculated and included in the report.
-If the variables could be assumed to follow a normal distribution, the Dunnett many-one t-test, based on a pooled variance estimate, was used for inter-group comparisons (i.e. single treatment groups against the control group).
-The Steel test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
-Fisher's Exact test for 2x2 tables was applied if the variable could be dichotomized without loss of information.

Indices:

N/A

Historical control data:

Historical control data was provided in the report and comparisons for certain parameters are described in the results section below.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
See remarks on results section.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
See remarks on results section.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were noted at any of the administered doses

Remarks on result:

other: Developmental toxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Analysis of Dose Formulations:

Investigations on concentrations, homogeneity, and stability in the used formulations were conducted by a method as supplied by the Sponsor. The measured values were within the required ranges (99.0 -111.0 % and 96.2 -104.6 % of recovery for the first and the second sampling, respectively).

Maternal Data

1. Mortality- 2 animals in the 1000 mg/kg bw/day dose group died prior to scheduled necropsy. One of the dams, which died on day 20 post coitum, showed an abdominal cavity with bluish stained intestinal organs. In the thoracic cavity, the thymus was bluish stained (about 3 hours after the administration of the test substance). Therefore, it was considered most likely due to an intubation error on day 20 post coitum.

The other dam was found dead in the morning of day 17 post coitum. During that necropsy, the dam was noted with bluish colored organs in the abdominal and thoracic cavities that were already autolytic. Including the dam's clinical signs of days 15 and 16 post coitum (rales, apathy and ventral recumbency), it was considered most likely due to an intubation error on day 15 post coitum. All other dams survived until scheduled necropsy.

2. Clinical signs- In the 1000 mg/kg bw/day group, the dams were noted without any clinical signs until day 7 post coitum. Starting on days 8 to 10 post coitum, bluish stained bedding material and dark colored feces were noted for all animals. As mentioned before, the dam that died on day 17 post coitum was seen with rales on days 15 and 16 post coitum and ventral recumbency on day 16 post coitum. On day 17 post coitum it was found dead in the cage. No clinical symptoms were noted in the dam found dead on day 20 post coitum.

In the 300 mg/kg bw/day group, the dams were noted without any clinical signs until day 7 post coitum. Starting on days 8 to 10 post coitum, dark colored feces were noted for all animals.

In the 100 mg/kg bw/day group, the dams were noted without any clinical signs until day 8 post coitum. Starting on days 9 to 11 post coitum, dark colored feces were noted for all animals. One dam was noted with hair loss at the lumbar and sacral region.

The discoloration on the bedding in the high dose group and the discolored feces in all test substance group were considered to be due to the staining property of the test substance. No clinical signs were noted for the vehicle control group.

3. Food consumption- No test substance related effects on food consumption occurred.

4. Body weights- No test substance related effect on body weight and body weight gain occurred. A statistically significant increase in mean body weight gain was noted in the 300 mg/kg bw/day and 1000 mg/kg bw/day groups during on days 8 and 9 post coitum and days 8, 9, 10 and 11 post coitum, respectively. This was considered incidental as mean body weight and mean food consumption of the 300 mg/kg bw/day and 1000 mg/kg bw/day dose groups did not correspond with it. For all other days, the mean body weight gain in the 300 mg/kg bw/day and 1000 mg/kg bw/day dose groups was similar to that of the control group. The mean body weight gain in the 100 mg/kg bw/day dose group was similar to that of the control group during the study.

5. Reproduction data- Relevant reproduction parameters (post implantation loss, number of fetuses, number of embryonic/fetal resorptions, number of abnormal fetus at external examination) were not influenced by treatment with the test substance.

Statistically significant lower numbers of post-implantation loss (% of implantation sites: 2.7 and 2.5, respectively) and embryonic/fetal resorptions (total: 8 and 7 %, respectively) in the 100 mg/kg bw/day and 300 mg/kg bw/day were considered to reflect the range of biological variation (the range of historical data shows a post implantation loss in % of 1.1 -12.2, and the embryonic/fetal resorptions as total between 3 -37).

6. Necropsy findings- All mated females were pregnant except three (2 from the 100 mg/kg bw/day group and 1 from the 300 mg/kg bw/day group).

During the two un-scheduled thorough necropsies, the dam that died on day 20 post coitum from the 1000 mg/kg bw/day group showed an abdominal cavity with bluish stained intestinal organs. In the thoracic cavity, the thymus was bluish stained (about 3 hours after the administration of the test substance). The other dam from this group which was found dead on day 17 post coitum was noted with bluish stained organs in the abdominal and thoracic cavities that were already autolytic. The findings of these two dams were considered due to an intubation error leading to colored abdominal organs and to colored abdominal and thoracic organs, respectively.

As mainly in all test substance administered dams, only the intestinal tract was observed with dose dependently increasing bluish discolorations, theses findings were considered to be due to the staining properties of the test substance.

In the 1000 mg/kg bw/day group, stomach (10/20) dams), small intestine (10/20 dams), caecum (20/20 dams) and colon (20/20 dams) were bluish colored. All other organs were observed without any abnormal findings at necropsy.

In the 300 mg/kg bw/day group, stomach (6/21 dams), small intestine (11/21 dams), caecum (21/21 dams) and colon (18/21 dams) were bluish colored. All other organs were observed without any abnormal findings at necropsy.

In the 100 mg/kg bw/day group, stomach (1/20 dams), small intestine (5/20 dams), caecum (3/20 dams) and colon (1/20 dams) were bluish colored. All other organs were observed without any abnormal findings at necropsy.

The dams in the control group were observed without any abnormal findings at necropsy.

 

Fetal Data

1. Body Weights- Mean fetal weights were considered to be not affected by treatment with the test substance. On litter basis, only the female fetuses of the 1000 mg/kg bw/day group were noted with a statistically significant higher mean body weight. On an individual basis, the female fetuses of the 100 mg/kg bw/day group, and the male and female fetuses of the 1000 mg/kg bw/day group were noted with a statistically significant higher mean body weight that included the change of statistically significant mean fetal weights on all fetuses in the 100 and 1000 mg/kg bw/day group. As these differences occurred without dose-dependency, the values were considered to reflect the normal range of biological variation (even more in combination with the total number of fetuses in each group.

2. Sex ratios- No test substance effects on sex ratio of the fetuses were noted in any group. Sex rations were close to 50 % in all groups with the following values in % (male/female): 47/53, 45/55, 50/50, and 50/50 in dose groups 0, 100, 300 and 1000 mg/kg bw/day, respectively.

3. External examination- External examinations of the fetuses did not reveal any test substance related findings in any group.

4. Visceral examination (microdissection technique)- Visceral examinations of the fetuses did not reveal any test substance related findings. During visceral examination of fixed fetuses findings were noted in:

61 out of 145 examined fetuses (in 20/22 litters) in the control group

48 out of 131 examined fetuses (in 18/20 litters) in the 100 mg/kg bw/day group

50 out of 145 examined fetuses (in 21/21 litters) in the 300 mg/kg bw/day group

49 out of 124 examined fetuses (in 20/20 litters) in the 1000 mg/kg bw/day group

Major findings (such as situs inversus, stomach malrotated, kidney and ureter absent, or renal pelvis/ureter severely dilated) were noted in the control, 300 mg/kg bw/day and 1000 mg/kg bw/day groups. These abnormalities were considered as spontaneous findings as no dose-dependency could be observed. The abnormality of a situs inversus (one control group fetus and one 300 mg/kg bw/day group fetus) and a malrotated stomach (one control group fetus) was covered by the actual historical control data. The findings of an absent kidney/ureter could not be covered by the actual historical data where only misshapen kidneys were noted. The control fetus with situs inversus showed a dilated renal pelvis with absent papilla and severely dilated renal ureter (right). Two fetuses (2 litters) from the control group only showed a dilated renal pelvis. This minor finding of a dilated renal pelvis was also noted in 4 fetuses (4 litters) from the 1000 mg/kg bw/day group. The historical control data also note 0 -1 fetus with dilated renal pelvis. In this study, about all noted fetuses with a dilated renal pelvis weighted less than the mean of litter mates, which may indicate some growth retardation.

In combination with these minor findings (renal pelvis dilated), the total number of kidney observations at the high dose (5 fetuses in 5 liters) was higher than in the control (3 fetuses in 3 litters). However, the minor finding of dilated renal pelvis in four fetuses and the single major abnormality of an absent kidney/ureter, with severe dilation of the remaining renal pelvis/ureter (one fetus from the 1000 mg/kg bw/day group), were considered unlikely to be of similar etiology. Therefore, in absence of other treatment related soft tissue changes, this slight difference from control was considered likely to be incidental.

5. Skeletal examination of fetuses (abnormal findings and common variants)- Skeletal examinations of the fetuses for abnormal findings and common variants did not reveal any test substance related findings. During skeletal examination of the fetuses abnormal findings were noted in

14 out of 133 fetuses (in 8/22 litters) in the control group

14 out of 120 fetuses (in 10/20 litters) in the 100 mg/kg bw/day group

11 out of 129 fetuses (in 8/21 litters) in the 300 mg/kg bw/day group

16 out of 114 fetuses (in 9/19 litters) in the 1000 mg/kg bw/day group

Two fetuses (2 litters) in the 100 mg/kg bw/day group were noted with more than three abnormalities (combined skeletal and cartilage observation). These multiple abnormities were considered as spontaneous findings as these fetuses weighed less than the mean of litter mates and no dose-dependency could be observed.

All other findings noted (like fused zygomatic arches, misshapen scapula, wavy ribs, fused thoracic vertebral arches, offset or bipartite ossified sternebrae, incompletely ossified bones of the cranium, non-ossified cervical vertebral bodies, incompletely and or non-ossified sternebrae, supernumerary and/or rudimentary ribs, non-ossified digits at fore limb and non-ossified toes, metatarsalia and talus at hind limb) also occurred without any indication of dose-relationship and about all were covered by the historical control data. As they also were commonly observed in this strain of rat, these findings were considered to be unrelated to the treatment with the test substance.

6. Skeletal examination of fetuses (stage of development)-Skeletal examinations of the fetuses for the stage of development did not reveal any test substance related findings. The noted, partly statistically significant incidences of more or less incompletely or non-ossified boned during skeletal examination revealed different stages of development with the emphasis that the fetuses of the 1000 mg/kg bw/day group were more developed than the fetuses of the control group. For some parameters (f.i. non-ossified cervical vertebral body 1, non-ossified digit 2 proximal phalanx left and right), the control group (on litter and on individual basis) was not covered by the historical control data. In some cases no group was covered by historical data (f.i. non-ossified toe 5 proximal phalanx left and right). As a general aspect these findings did not indicate any dose-dependency. Therefore these findings were considered to be unrelated to the treatment with the test substance.

7. Cartilage examination of fetuses (abnormal findings and common variants)- Cartilage examinations of the fetuses did not reveal any test substance related findings. During cartilage examination of the fetuses abnormal findings were noted in:

1 out of 133 fetuses (in 1/22 litters ) in the control group

2 out of 120 fetuses (in 2/20 litters) in the 100 mg/kg bw/day group

5 out of 129 fetuses (in 4/21 litters) in the 300 mg/kg bw/day group

3 out of 114 fetuses (in 3/19 litters) in the 1000 mg/kg bw/day group

One fetus in the 100 mg/kg bw/day group was noted with more than three findings (combined skeletal and cartilage observations). These multiple malformations were considered as spontaneous findings as there was no dose-dependency observed.

All other abnormal findings noted (long ventral plates, branched xiphoid cartilage or xiphoid cartilage with hole, long or interrupted costal cartilages 10, 11 uni/bilateral) also occurred without any indication of dose-relationship. Few incidences that were not covered by historical data were also noted without any dose-dependency. But they are commonly observed in this strain or rat. Therefore these findings were considered to be unrelated to the treatment with the test substance.

The noted, partly statistically significant common variants during cartilage examination did not indicate any dose relationships. Therefore these findings were considered to be unrelated to the treatment with the test substance.

Applicant's summary and conclusion

Conclusions:

The purpose of this study was to assess the effects of B119 HC Blue 16 on pregnant female rats and embryo-fetal development when administered orally, by gavage, once daily to mated female rats from day 6 through to day 20 post coitum, inclusive. Up to and including the high dose of 1000 mg/kg bw/day, administration of the test substance did not result in any signs of general or teratogenic toxicity. Therefore, a NOAEL for maternal and embryo-fetal effects was established at the high dose level of greater than 1000 mg/kg bw/day.

Executive summary:

The purpose of this study was to assess the effects of B119 HC Blue 16 on pregnant female rats and embryo-fetal development when administered orally, by gavage, once daily to mated female rats from day 6 through to day 20 post coitum, inclusive.

Each group consisted of 22 mated female rats. The test substance was administered once daily at dose levels of:

Group 1 -0 mg/kg bw/day (vehicle control)

Group 2 -100mg/kg bw/day

Group 3 -300mg/kg bw/day

Group 4 -1000 mg/kg bw/day

A standard dose volume of 10 mL/kg bw with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (highly purified water).

Test solutions were prepared daily. Samples of the formulations were taken before first administration and analyzed for concentrations, homogeneity, and stability (after 4 hours and 7 days) of the test substance and during the last week of administration for analyzing concentrations and homogeneity of the test substance.

Food consumption and body weight data were calculated for the periods from day 0 to 3, 3 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18 and 18 to 21 of gestation.

All surviving mated females were sacrificed on day 21 post coitum by CO2 -asphyxiation. A complete autopsy and a macroscopic examination of the organs were carried out. Examination of dams and fetuses was performed in accordance with international recommendations.

The fetuses were removed by Caesarean section. The uterine weight including the fetuses was determined to enable the calculation of the corrected body weight gain for the dams. The intact uterus was examined for the presence of resorption sites (early, late) and fetuses (live or dead). Their uterine position was recorded. The number of corpora lutea was also determined.

Each viable fetus was weighed, sexed and examined for gross external malformations and euthanized. The, half of the fetuses were prepared for the microdissection technique with fixation in Bouin's fixative. The other half of fetuses were eviscerated, processed for the cartilage (Alcian blue) and skeletal (Alizarin red S) staining and preserved for storage.

In case of non-pregnancy the number of implantation sites was recorded after staining the uterus in an aqueous solution of ammonium sulfide. The corpora lutea were counted.

Investigations on concentrations, homogeneity, and stability in the used formations were conducted by a method as supplied by the Sponsor. The measured values were within the required ranges (96.2 -111.0 % of recovery).

All animals except two dams in group 4 survived until scheduled necropsy. One dam that died on day 17 post coitum, was noted with rales two days prior to and with ventral recumbency one day prior to death. The necropsy revealed that all organs were bluish stained and already autolytic. The other dam that died pre-scheduled about 3 hours after administration on day 20 post coitum did not show any signs prior to death. Necropsy revealed that all intestinal organs were bluish stained as well as the thymus. Thus, the death of both animals was considered incidental due to misadministration on days 15 and 20 post coitum, respectively.

No test substance related signs of discomfort were noted in groups 2, 3 and 4. Up to and including 1000 mg/kg bw/day, food consumption and body weight development in the dams gave no indication of a test substance related effect.

As scheduled necropsy no test substance related adverse macroscopic findings were noted. In all groups all animals were noted with bluish discolored stomachs, and bluish discolored small and large intestines. The number of animals with bluish discolored abdominal organs increased according to the higher dose of groups 2, 3 and 4. These findings were considered related to the staining properties of the test substance.

The relevant reproduction data (incidence of post-implantation loss and number of fetuses per dam) were not affected by treatment with the test substance.

Sex ratio values in % (male/female) were 47/53, 45/55, 50/50 and 50/50 in groups 1, 2, 3, and 4, respectively.

Mean fetal weights in groups 2, 3 and 4 were not influenced by treatment with the test substance. External, visceral, and skeletal examinations of fetuses at Cesarean necropsy did not reveal any test substance related findings.

In conclusion, up to and including the high dose of 1000 mg/kg bw/day, administration of the test substance did not result in any signs of general or teratogenic toxicity. Therefore, a NOAEL for maternal and embryo-fetal effects was established at being greater than the high dose level of 1000 mg/kg bw/day.